Bacteria Cultures and Compositions Comprising Bacteria Cultures

ABSTRACT

The present invention relates to bacteria cultures and composition comprising one or more cultures of the invention. The invention also relates to methods of washing or cleaning laundry or fabrics and surfaces as well as degrading waste material using a bacteria culture of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/751,411 filed Jan. 28, 2013 (now allowed), which is a continuation ofU.S. application no. 12/374,361 filed Jan. 19, 2009 (now U.S. Pat. No.8,383,097) which is a 35 U.S.C. 371 national application ofPCT/US2007/075185 filed Aug. 3, 2007, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application Nos.60/837,065 and 60/891,279 filed Aug. 11, 2006 and Feb. 23, 2007,respectively, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to isolated bacteria cultures andcompositions comprising said cultures. A composition of the inventionmay advantageously be used for washing, especially laundry or newlymanufactured fabrics, cleaning of surfaces, such as carpets, cleaning ofdrains and septic tanks, as well as degrading waste material.

BACKGROUND

Compositions for washing laundry often contain surfactants and otheractive ingredients such as enzymes for removing stubborn stains. Enzymesmay not be able to remove all kinds of complex strains.

WO 03/099987 discloses an article and method of cleaning fabric, whereinsoiled fabric is soaked in water in the presence of an articlecontaining one or more harmless microorganisms capable of excretingenzymes useful for cleaning.

Even though a huge number of composition and cleaning systems are knownin the art there is never the less still a desire for compositions whichexhibit strong washing and cleaning capabilities. There is still acontinuing need for providing efficient compositions for washing andcleaning of laundry, fabrics and surfaces.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising selected wholebacteria cultures. The bacteria are isolated from their naturalenvironment. The composition of the invention may be used for washingespecially laundry and newly manufactured fabrics and cleaning surfacessuch as carpets. A composition of the invention may optionally besupplemented with surfactants and/or other active ingredients, such asenzymes.

It has been found that selected (whole) bacteria cultures of theinvention have washing and cleaning benefits when used for washinglaundry and fabrics and/or cleaning surfaces. More specifically theinventors found that the bacteria cultures of the invention derived fromstrains of the genus Bacillus, preferably strains of the speciesBacillus subtilis, Bacillus amyloliquefaciens, Bacillus simplex,Bacillus velezensis, and Bacillus atrophaeus, and compositionscontaining one or more bacteria cultures of the invention, have strongwash performance and cleaning efficacy.

In the first aspect the invention relates to bacteria cultures havingcharacteristics substantially identical to that of a strain selectedfrom the group consisting of:

the strain having the deposit accession number PTA-7541;

the strain having the deposit accession number PTA-7542;

the strain having the deposit accession number PTA-7543;

the strain having the deposit accession number PTA-7544;

the strain having the deposit accession number PTA-7545;

the strain having the deposit accession number PTA-7546;

the strain having the deposit accession number PTA-7547;

the strain having the deposit accession number PTA-7548;

the strain having the deposit accession number PTA-7549.

the strain having the deposit accession number PTA-7550,

the strain having the deposit accession number PTA-7789,

the strain having the deposit accession number PTA-7790,

the strain having the deposit accession number PTA-7791,

the strain having the deposit accession number PTA-7792,

the strain having the deposit accession number PTA-7793, or, a mixtureof two or more of the strains.

In a second aspect the invention relates to a composition comprising oneor more biologically active cultures of the invention. In a preferredembodiment the composition also contains one or more ingredients,including surfactants, hydrotropes, preservatives, fillers, builders,stabilizer, fragrances, anti-redeposition agents, nutrients,biostimulants, and enzymes; or a combination of one or more thereof.

In other aspects the invention relates to methods of washing andcleaning fabrics and surfaces, respectively, comprising subjecting saidobjects to a bacteria culture or composition of the invention. Theinvention also relates to degrading waste material using a culture orcomposition of the invention.

In context of the invention soils/stains especially contemplated includeblood, butterfat, cooking oil, sebum, and ballast. The term “ballast” isan art-recognized term for a general “soil” containing chocolate, blood,red wine, and milk mixed together. Other soils/stains contemplatedinclude (e.g., pork) lard, (e.g., hamburger) oil, (e.g., hamburger)grease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated (whole) bacteria cultures andcompositions comprising one or more of said cultures. The cultures andcompositions may be used for various washing and cleaning purposes,especially laundry and fabric washing as well as surface cleaning. Otheruses, including waste degradation, are also contemplated.

Cultures of the Invention

In the first aspect the invention relates to bacteria cultures havingcharacteristics substantially identical to that of a strain selectedfrom the group consisting of:

the strain having the deposit accession number PTA-7541;

the strain having the deposit accession number PTA-7542;

the strain having the deposit accession number PTA-7543;

the strain having the deposit accession number PTA-7544;

the strain having the deposit accession number PTA-7545;

the strain having the deposit accession number PTA-7546;

the strain having the deposit accession number PTA-7547;

the strain having the deposit accession number PTA-7548;

the strain having the deposit accession number PTA-7549.

the strain having the deposit accession number PTA-7550,

the strain having the deposit accession number PTA-7789,

the strain having the deposit accession number PTA-7790,

the strain having the deposit accession number PTA-7791,

the strain having the deposit accession number PTA-7792,

the strain having the deposit accession number PTA-7793, or, a mixtureof two or more of the strains.

In a preferred embodiment a culture of the invention has propertiesidentical to one of above mentioned deposited strains, or a mixturethereof. The culture may preferably be one or more of the abovementioned deposited strains. A culture of the invention may be a progenyof one of the deposited strains. A culture of the invention ispreferably substantially pure, such as at least 90% pure, preferably atleast 95% pure, more preferably at least 97% pure, even more preferablyat least 99% pure.

The deposited bacteria cultures are derived from isolated naturallyoccurring bacteria strains. All strains were collected in the UnitedStates in 2005. Cultures of the invention may consist of dormantbacteria spores and/or viable bacteria.

A culture of the invention having substantially identicalcharacteristics of one or more of the deposited strains may be derivedfrom any bacteria, preferably from strains of the genus Bacillus,especially strains derived from species selected from the groupconsisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillussimplex, Bacillus velezensis, and Bacillus atrophaeus.

Above mentioned deposited strains were deposited on 20 Apr. 2006 and 18Aug. 2006, as indicated in more details below in the “Materials &Methods”-section, under terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108, USA.

In embodiments of the invention two or more bacteria cultures of theinvention are combined. Preferred combinations include deposited strainsPTA-7547 and PTA-7548 which, as indicated below, are suitable forsurface cleaning, especially carpet cleaning.

Composition of the Invention

In the second aspect the invention relates to a composition comprisingone or more cultures of the invention.

A composition of the invention has a number of potential advantages overfor instance, traditional enzymatic washing and/or cleaning compositionsas, e.g., laundry and/or other soiled objects with complex and/orstubborn stains in general require multi-enzyme washing or cleaningsystems. Compositions of the invention comprise one or more bacteriacultures of the invention having at their disposal the entire metabolicpotential of the bacteria culture, or a combination of one or morecultures. Due to the cost of preparing effective multi-enzymecompositions, adding a bacteria culture as an active stain removingingredient may be a good and/or cost efficient alternative tocompositions comprising, e.g., mono-component enzymes. A bacterialculture of the invention may also advantageously be used to at leastpartly substitute enzymes in washing or cleaning compositions. In anembodiment the composition comprises from 0.1-90 wt-% culture,preferably 0.5-50 wt.-% culture, especially from 1-10 wt-% culture ofthe invention.

In a preferred embodiment a composition of the invention also containsone or more surfactants and/or optionally other active ingredients, suchas enzymes. A composition of the invention may be in solid or liquidform. The composition may be a concentrate to be diluted, rehydratedand/or dissolved in a solvent, including water, before use. Thecomposition may also be a ready-to-use (in-use) composition. Thecomposition may furthermore be an active cleaning base ingredient to beincorporated into other cleaning or washing compositions.

Other contemplated ingredients include surfactants, hydrotropes,preservatives, fillers, builders, complexing agents, polymers,stabilizers, perfumes, biostimulants or nutrients, conventionaldetergent ingredients, and enzymes, or combinations of one or morethereof.

Surfactants

The surfactants may be non-ionic including semi-polar and/or anionicand/or cationic and/or zwitterionic. The surfactant(s) should cause aslittle harm to the bacteria culture's activity as possible.

The surfactants may be present in a composition of the invention at alevel of from 0.1% to 60% by weight.

In one embodiment the composition contains from about 1% to about 40% ofan anionic surfactant such as linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylester, alkyl- or alkenylsuccinic acid or soap.

In an embodiment the composition contains from about 0.2% to about 40%of a non-ionic surfactant such as alcohol ethoxylate, nonylphenolethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylatedfatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxyalkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine(“glucamides”).

Hydrotropes

The composition may contain hydrotropes. The term “hydrotrope” generallymeans a compound with the ability to increase the solubilities,preferably aqueous solubilities, of certain slightly soluble organiccompounds. Examples of hydrotropes include sodium xylene sulfonate (SXS)and sodium cumene sulfonate (SCS).

Metal Chelation Agents

The composition may contain a metal chelating agent such as carbonates,bicarbonates, and sesquicarbonates.

Solvents

The composition may comprise a solvent such as water or an organicsolvent such as isopropyl alcohol or a glycol ether.

Builders or Complexing Agents

The composition may also contain 0-65% of a builder or complexing agentsuch as zeolite, phosphates, such as diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, silicates, such as soluble silicates,metasilicates, layered silicates (e.g. SKS-6 from Hoechst).

Polymers

The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

Stabilizers

If an enzyme(s) is(are) present in the composition it(they) may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

Detergent Ingredients

The composition may also contain other conventional detergentingredients such as, e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil redeposition agents, dyes, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

In an embodiment the solid composition contains the followingconstitutes: hydrotropes, anionic or nonionic surfactants, builders,carbonates for pH control and metal chelation, solvents, fillers, dye,perfume, and fluorescent whitening agent.

Cleaning Compositions Suitable for Surface Cleaning

The bacterial cultures of the invention may be used in a compositionsuitable for cleaning surfaces, such as hard and soft surfaces, such asespecially carpets and the like. Examples of hard and soft surfaces arementioned below.

In a preferred embodiment a bacteria culture of the invention or acombination of two or more cultures are used in a surface cleaningcomposition comprising a surfactant system or cleaning composition. In apreferred embodiment the composition is a carpet cleaner composition,i.e., a carpet cleaning composition comprising a surfactant system orcleaning composition, e.g., a surfactant system or cleaning compositiondisclosed in WO 2007/076337 (which is hereby incorporated by reference).The carpet cleaner may be a carpet extraction cleaner or a carpet spotremover.

In one embodiment said surfactant system comprises two or more nonionicsurfactants and an anionic surfactant. In an embodiment one of thenonionic surfactants is a water insoluble nonionic surfactant. Further,in another embodiment the surfactant system comprises two or more watersoluble nonionic surfactants and one water insoluble nonionicsurfactant. Further, the surfactant system may also comprise one watersoluble anionic surfactant, one water-soluble nonionic surfactant andone water insoluble nonionic surfactant.

The ratio between anionic surfactant(s) and nonionic surfactant(s) mayin a preferred embodiment be between 10:1 and 1:10, preferably between10:1 and 1:1, more preferably between 8:1 and 1:1, even more preferablybetween 6:1 and 1:1.

In an embodiment of the invention the cleaning composition is formulatedas follows:

COMPONENT PERCENT BY WEIGHT Solvent  50-95 Anionic surfactant. 2.5-15Water insoluble nonionic surfactant 2.5-15 Buffer salts 0.25-1  BacteriaCulture of the invention 10⁵-10⁹ cfu/ml cleaning composition Optionallyother ingredients 0.1-10

The surfactants (including ratio between surfactants), solvents, salts,and optional ingredients (such as enzymes) may be any mentioned above orbelow.

Anionic Surfactants

The anionic surfactant(s) may be water soluble anionic surfactantsand/or water insoluble anionic surfactants. Water soluble anionicsurfactants are preferred.

Examples of suitable water soluble anionic surfactants include those toselected from the group consisting of alkyl sulfates, alkyl ethersulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates,alkyl aryl sulfates, alkyl aryl sulfonates, monoglyceride sulfates,alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, benzenesulfonates, toluene sulfonates, xylene sulfonates, cumene sulfonates,alkyl benzene sulfonates, alkyl diphenyloxide sulfonate, alpha-olefinsulfonates, alkyl naphthalene sulfonates, paraffin sulfonates, ligninsulfonates, alkyl sulfosuccinates, ethoxylated sulfosuccinates, alkylether sulfosuccinates, alkylamide sulfosuccinates, alkylsulfosuccinamate, alkyl sulfoacetates, alkyl phosphates, phosphateester, alkyl ether phosphates, acyl sarconsinates, acyl isethionates,N-acyl taurates, N-acyl-N-alkyltaurates, and alkyl carboxylates.

Examples of preferred water soluble anionic surfactants include sodiumdodecyl sulfate (sodium lauryl sulfate), sodium laureth sulfate (sodiumlauryl ether sulfate), sodium dodecyl benzene sulfonate, disodium octylsulfosuccinate, sodium butyl naphthalene sulfonate, ethoxylated sodiumlauryl sulfosuccinate, sodium stearate, and sodium lauroyl sarcoside, ora mixture of two or more. Examples of anionic surfactants are alsomentioned in WO 2007/076337 (see page 7, line 8 to page 9, line 3—whichis hereby incorporated by reference).

Non-Ionic Surfactants

The surfactant system may contain a non-ionic surfactant. The nonionicsurfactant may preferably be a water insoluble nonionic surfactant or awater soluble nonionic surfactant, or mixtures thereof. Examples ofsuitable nonionic surfactants are given below.

Examples of suitable water insoluble nonionic surfactants include alkyland aryl: glycerol ethers, glycol ethers, ethanolamides,sulfoanylamides, alcohols, amides, alcohol ethoxylates, glycerol esters,glycol esters, ethoxylates of glycerol ester and glycol esters,sugar-based alkyl polyglycosides, polyoxyethylenated fatty acids,alkanolamine condensates, alkanolamides, tertiary acetylenic glycols,polyoxyethylenated mercaptans, carboxylic acid esters, andpolyoxyethylenated polyoxyproylene glycols. Also included are EO/POblock copolymers (EO is ethylene oxide, PO is propylene oxide), EOpolymers and copolymers, polyamines, and polyvinylpynolidones.

Water soluble nonionic surfactants typically have a higher ethyleneoxide content in the hydrophilic region of the surfactant in comparisonto water insoluble nonionic surfactants.

In an embodiment the water soluble nonionic surfactant is a linearprimary, or secondary or branched alcohol ethoxylate having the formula:RO(CH₂CH₂O)_(n)H, wherein R is the hydrocarbon chain length and n is theaverage number of moles of ethylene oxide. In a preferred embodiment Ris linear primary or branched secondary hydrocarbon chain length in therange from C9 to C16 and n ranges from 6 to 13. Especially preferred isthe alcohol ethoxylate where R is linear C9-C11 hydrocarbon chainlength, and n is 6.

Examples of commercially available water soluble nonionic alcoholethoxylate surfactants include NEODOL™ 91-6, TOMADOL™ 91-6, orBIO-SOFTT™ N23-6.5.

Examples of non-ionic surfactants are also mentioned in WO 2007/076337(see page 9, line 5 to page 12, line 14—which is hereby incorporated byreference).

Examples of specific carpet cleaner compositions are disclosed inExamples 10 and 11 below. Any bacteria culture of the invention, orcombinations thereof, may be used. However, in a preferred embodimentthe bacteria cultures used are PTA-7548 and PTA-7547. The ratio betweenthe two cultures may be between 1:10 to 10:1, preferably 1:2 to 2:1,such as around 1:1.

The bacterial culture(s) of the invention should be present in thecleaning composition, such as carpet cleaners, in effective amounts.Effective amounts can to easily be determined by one skilled in the art.

Salts and Buffer Salts

The cleaning composition may contain one or more salts and/or buffersalts. The salts or buffer salts may be any known inorganic salt, but ispreferably a salt selected from the group consisting of alkali metalsalts of nitrates, acetates, chlorides, bromides, iodides, sulfates,hydroxides, carbonates, hydrogen carbonates, (also called bicarbonates),phosphates, sulfides, and sulfites; ammonium salts of nitrates,acetates, chlorides, bromides, iodides, sulfates, hydroxides,carbonates, hydrogen carbonates (also called bicarbonates), phosphates,sulfides, and sulfites; alkaline earth metal salts of nitrates,chlorides, bromides, iodides, sulfates, sulfides, and hydrogencarbonates; manganese, iron, copper, and zinc salts of nitrates,acetates, chlorides, bromides, iodides, and sulfates; citrates andborates.

Especially contemplated are carbonates or bicarbonates, in particularselected from the group consisting of sodium carbonate and sodiumbicarbonate, or a mixture thereof. In a specific embodiment the ratiobetween sodium carbonate and sodium bicarbonate is between 1:10 to 10:1.

The total amount of salts and/or buffer salts is preferably between 0.8to 8 wt. %, preferably 1-5 wt. %, more preferably around 2 wt. % of thefinal in-use cleaning composition.

Enzymes

One or more enzyme activities may be present in a composition of theinvention and when practicing a method of the invention. Especiallycontemplated enzymes include proteases, alpha-amylases, cellulases,lipases, peroxidases/oxidases, pectate lyases, and mannanases, ormixtures thereof.

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metallo protease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g. of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274. Preferred commercially available protease enzymes includeALCALASE™, SAVINASE™, PRIMASE™, DURALASE™, DYRAZYM™, ESPERASE™,EVERLASE™, POLARZYME™, KANNASE™, LIQUANASE™ (Novozymes A/S), MAXATASET™,MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™, FN2™, and FN3™(Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis(Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360),B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ ANDLIPOLASE ULTRA™, or LIPEX™ (Novozymes A/S).

Cutinase:

The method of the invention may be carried out in the presence ofcutinase. classified in EC 3.1.1.74.

The cutinase used according to the invention may be of any origin.Preferably cutinases are of microbial origin, in particular ofbacterial, of fungal or of yeast origin.

Cutinases are enzymes which are able to degrade cutin. In a preferredembodiment, the cutinase is derived from a strain of Aspergillus, inparticular Aspergillus oryzae, a strain of Alternaria, in particularAlternaria brassiciola, a strain of Fusarium, in particular Fusariumsolani, Fusarium solani pisi, Fusarium roseum culmorum, or Fusariumroseum sambucium, a strain of Helminthosporum, in particularHelminthosporum sativum, a strain of Humicola, in particular Humicolainsolens, a strain of Pseudomonas, in particular Pseudomonas mendocina,or Pseudomonas putida, a strain of Rhizoctonia, in particularRhizoctonia solani, a strain of Streptomyces, in particular Streptomycesscabies, or a strain of Ulocladium, in particular Ulocladiumconsortiale. In a most preferred embodiment the cutinase is derived froma strain of Humicola insolens, in particular the strain Humicolainsolens DSM 1800. Humicola insolens cutinase is described in WO96/13580 which is herby incorporated by reference. The cutinase may be avariant, such as one of the variants disclosed in WO 00/34450 and WO01/92502, which are hereby incorporated by reference. Preferred cutinasevariants include variants listed in Example 2 of WO 01/92502, which ishereby specifically incorporated by reference.

Preferred commercial cutinases include NOVOZYM™ 51032 (available fromNovozymes A/S, Denmark).

The method of the invention may be carried out in the presence ofphospholipase classified as EC 3.1.1.4 and/or EC 3.1.1.32. As usedherein, the term phospholipase is an enzyme which has activity towardsphospholipids. Phospholipids, such as lecithin or phosphatidylcholine,consist of glycerol esterified with two fatty acids in an outer (sn-1)and the middle (sn-2) positions and esterified with phosphoric acid inthe third position; the phosphoric acid, in turn, may be esterified toan amino-alcohol. Phospholipases are enzymes which participate in thehydrolysis of phospholipids. Several types of phospholipase activity canbe distinguished, including phospholipases A₁ and A₂ which hydrolyze onefatty acyl group (in the sn-1 and sn-2 position, respectively) to formlysophospholipid; and lysophospholipase (or phospholipase B) which canhydrolyze the remaining fatty acyl group in lysophospholipid.Phospholipase C and phospholipase D (phosphodiesterases) release diacylglycerol or phosphatidic acid respectively.

The term phospholipase includes enzymes with phospholipase activity,e.g., phospholipase A (A₁ or A₂), phospholipase B activity,phospholipase C activity or phospholipase D activity. The term“phospholipase A” used herein in connection with an enzyme of theinvention is intended to cover an enzyme with Phospholipase A₁ and/orPhospholipase A₂ activity. The phospholipase activity may be provided byenzymes having other activities as well, such as, e.g., a lipase withphospholipase activity. The phospholipase activity may, e.g., be from alipase with phospholipase side activity. In other embodiments of theinvention the phospholipase enzyme activity is provided by an enzymehaving essentially only phospholipase activity and wherein thephospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, e.g., of animal origin (such as,e.g., mammalian), e.g. from pancreas (e.g., bovine or porcine pancreas),or snake venom or bee venom. Preferably the phospholipase may be ofmicrobial origin, e.g., from filamentous fungi, yeast or bacteria, suchas the genus or species Aspergillus, e.g., A. niger; Dictyostelium,e.g., D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M.subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g., R.pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer;Sclerotinia, e.g., S. libertiana; Trichophyton, e.g. T. rubrum;Whetzelinia, e.g., W. sclerotiorum; Bacillus, e.g., B. megaterium, B.subtilis; Citrobacter, e.g., C. freundii; Enterobacter, e.g., E.aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g., E.herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae;Proteus, e.g., P. vulgaris; Providencia, e.g., P. stuartii; Salmonella,e.g. S. typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens;Shigella, e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber;Yersinia, e.g., Y. enterocolitica. Thus, the phospholipase may befungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium,such as a strain of F. culmorum, F. heterosporum, F. solani, or a strainof F. oxysporum. The phospholipase may also be from a filamentous fungusstrain within the genus Aspergillus, such as a strain of Aspergillusawamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nigeror Aspergillus oryzae.

Preferred phospholipases are derived from a strain of Humicola,especially Humicola lanuginosa. The phospholipase may be a variant, suchas one of the variants disclosed in WO 00/32758, which are herebyincorporated by reference. Preferred phospholipase variants includevariants listed in Example 5 of WO 00/32758, which is herebyspecifically incorporated by reference. In another preferred embodimentthe phospholipase is one described in WO 04/111216, especially thevariants listed in the table in Example 1.

In another preferred embodiment the phospholipase is derived from astrain of Fusarium, especially Fusarium oxysporum. The phospholipase maybe the one concerned in WO 98/026057 displayed in SEQ ID NO: 2 derivedfrom Fusarium oxysporum DSM 2672, or variants thereof.

In a preferred embodiment of the invention the phospholipase is aphospholipase A₁ (EC. 3.1.1.32). In another preferred embodiment of theinvention the phospholipase is a phospholipase A₂ (EC.3.1.1.4.).

Examples of commercial phospholipases include LECITASE™ and LECITASE™ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S, Denmark).

Amylases:

Suitable amylases (alpha and/or beta) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, alpha-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839, or the Bacillus sp. strains disclosed in WO95/026397 or WO 00/060060.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, WO 97/43424, WO 01/066712, WO 02/010355, WO02/031124 and WO 2006/002643 (which references all incorporated byreference.

Commercially available amylases are DURAMYL™, TERMAMYL™, TERMAMYLULTRA™, NATALASE™, STAINZYME™, FUNGAMYL™ and BAN™ (Novozymes A/S),RAPIDASE™ and PURASTAR™ (from Genencor International Inc.).

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.

Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Thielavia terrestris, Myceliophthorathermophila, and Fusarium oxysporum disclosed in U.S. Pat. No.4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat.No. 5,776,757, WO 89/09259, WO 96/029397, and WO 98/012307.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include CELLUZYME™, CAREZYMET™,ENDOLASE™, RENOZYME™ (Novozymes A/S), CLAZINASE™ and PURADAX HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g. from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ and NOVOZYM™ 51004(Novozymes A/S).

Pectate Lyases (Also Called Polygalacturonate Lyases):

Examples of pectate lyases include pectate lyases that have been clonedfrom different bacterial genera such as Erwinia, Pseudomonas, Klebsiellaand Xanthomonas, as well as from Bacillus subtilis (Nasser et al. (1993)FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994)Biosci. Biotech. Biochem. 58:947-949). Purification of pectate lyaseswith maximum activity in the pH range of 8-10 produced by Bacilluspumilus (Dave and Vaughn (1971) J. Bacteriol. 108:166-174), B. polymyxa(Nagel and Vaughn (1961) Arch. Biochem. Biophys. 93:344-352), B.stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol.26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci.31:838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J.Microbiol. 24:1164-1172) have also been described. Any of the above, aswell as divalent cation-independent and/or thermostable pectate lyases,may be used in practicing the invention. In preferred embodiments, thepectate lyase comprises the amino acid sequence of a pectate lyasedisclosed in Heffron et al., (1995) Mol. Plant-Microbe Interact. 8:331-334 and Henrissat et al., (1995) Plant Physiol. 107: 963-976.Specifically contemplated pectate lyases are disclosed in WO 99/27083and WO 99/27084. Other specifically contemplated pectate lyases derivedfrom Bacillus licheniformis is disclosed as SEQ ID NO: 2 in U.S. Pat.No. 6,284,524 (which document is hereby incorporated by reference).Specifically contemplated pectate lyase variants are disclosed in WO02/006442, especially the variants disclosed in the Examples in WO02/006442 (which document is hereby incorporated by reference).

Examples of commercially available alkaline pectate lyases includeBIOPREP™ and SCOURZYME™ L from Novozymes A/, Denmark.

Mannanase:

Examples of mannanases (EC 3.2.1.78) include mannanases of bacterial andfungal origin. In a specific embodiment the mannanase is derived from astrain of the filamentous fungus genus Aspergillus, preferablyAspergillus niger or Aspergillus aculeatus (WO 94/25576). WO 93/24622discloses a mannanase isolated from Trichoderma reseei. Mannanases havealso been isolated from several bacteria, including Bacillus organisms.For example, Talbot et al., Appl. Environ. Microbiol., Vol. 56, No. 11,pp. 3505-3510 (1990) describes a beta-mannanase derived from Bacillusstearothermophilus. Mendoza et al., World J. Microbiol. Biotech., Vol.10, No. 5, pp. 551-555 (1994) describes a beta-mannanase derived fromBacillus subtilis. JP-A-03047076 discloses a beta-mannanase derived fromBacillus sp. JP-A-63056289 describes the production of an alkaline,thermostable beta-mannanase. JP-A-63036775 relates to the Bacillusmicroorganism FERM P-8856 which produces beta-mannanase andbeta-mannosidase. JP-A-08051975 discloses alkaline beta-mannanases fromalkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillusamyloliquefaciens is disclosed in WO 97/11164. WO 91/18974 describes ahemicellulase such as a glucanase, xylanase or mannanase active.Contemplated are the alkaline family 5 and 26 mannanases derived fromBacillus agaradhaerens, Bacillus licheniformis, Bacillus halodurans,Bacillus clausii, Bacillus sp., and Humicola insolens disclosed in WO99/64619. Especially contemplated are the Bacillus sp. mannanasesconcerned in the Examples in WO 99/64619 which document is herebyincorporated by reference.

Examples of commercially available mannanases include MANNAWAY™available from Novozymes A/S Denmark.

The enzyme(s) may be present in a composition of the invention is anamount from 0.1-10 wt-%, preferably 0.5-5 wt-%, especially 1-2 wt-% ofthe composition.

Method of the Invention

In the third aspect the invention relates to methods of washing laundryor fabrics comprising subjecting said laundry or fabric to a compositionor bacteria culture of the invention.

The method of the invention may be carried as out by adding acomposition or bacteria culture of the invention to washing liquor,which may or may not contain the laundry or fabric to be washed. It isimportant to insure proper conditions during washing or cleaning toallow the bacteria culture in question to degrade the soils/stains inquestion. In case dormant bacteria spores are used suitable conditionsand/or ingredients for germination may be required. It is important tounderstand that the storage condition for bacteria cultures orcompositions of the invention may be different from in-use conditions.

A method of washing laundry or fabric or cleaning surfaces of theinvention may be carried out as a one-step method or a two-step method.The treatment steps may be carried out simultaneously or sequentially.In one embodiment treatment is carried out using a culture and one ormore active ingredients (as described above) simultaneously. Accordingto the invention laundry or fabric may be treated with a bacteriaculture of the invention and one or more active ingredients sequentiallyin one or two baths. In an embodiment the method of the invention may becarried out in two steps, i.e., by first treating the laundry, fabric orsurface in question with a bacteria culture of the invention andsubsequently or simultaneously with an active ingredient, especiallyenzyme, e.g., a protease, alpha-amylase, cellulase, lipase,peroxidases/oxidase, pectate lyase, and mannanase, or mixtures thereof.A two step method of the invention may be carried out in one bath orsequentially in two (separate) baths.

The bacterial culture or composition of the invention is used in aneffective concentration during a method of the invention. In anembodiment the concentration of bacteria culture during washing may bein the range from 1×10⁶ to 1×10¹² bacteria cells per L wash liquor,preferably above 1×10⁷ bacteria cells per L wash liquor.

The pH during washing may be in the range from 5-11. The temperature maytypically be in the range from 10-90° C., preferably 20-50° C. In anembodiment washing is carried out for a period between 1 and 1440minutes. The fabric:wash liquor ratio may preferably in the range from1:1 to 1:20, preferably 1:10. As mentioned above one or more enzymes maybe present during washing. Contemplated enzymes include any of the onesmentioned in the “Enzymes” section above, which include proteases,alpha-amylases, cellulases, lipases, peroxidase/oxidase, mannanases,pectate lyases, or a mixture thereof. Enzymes may be present in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor,in particular 0.1-1 mg of enzyme protein per liter of wash liquor. In apreferred embodiment the laundry or fabric is rinsed after washing.

Laundry and/or Fabrics

When using the term “fabrics” it encompasses all kind of fabrics,textiles, fibers, clothes garments and the like. “Laundry” is, incontrast to “newly manufactured fabrics”, already used and/orstained/soiled clothes in need of washing. Washing laundry is typicallycarried out in private households, while washing newly manufacturedfabrics are mainly done in the textile industry. Washing of laundry canalso occur in commercial and institutional facilities such as hospitals,prisons, uniform service companies, and the like. The fabric or laundrymay be made from any suitable material. In preferred embodiments thefabrics and/or laundry are made from cellulosic materials, syntheticmaterials and/or man-made fibers, or blends thereof.

Examples of contemplated cellulosic materials include cotton, viscose,rayon, ramie, linen, lyocell (e.g., TENCEL™, produced by CourtauldsFibers), or blends thereof, or blends of any of these fibers togetherwith synthetic or man-made fibers (e.g., polyester, polyamid, nylon) orother natural fibers such as wool and silk, such as viscose/cottonblends, lyocell/cotton blends, viscose/wool blends, lyocell/wool blends,cotton/wool blends; flax (linen), ramie and other fabrics and/or laundrybased on cellulose fibers, including all blends of cellulosic fiberswith other fibers such as wool, polyamide, acrylic and polyester fibers,e.g., viscose/cotton/polyester blends, wool/cotton/polyester blends,flax/cotton blends etc. The fabric and/or laundry may also be asynthetic materials, e.g., consisting of essentially 100% polyester,polyamid, nylon, respectively. The term “wool,” means any commerciallyuseful animal hair product, for example, wool from sheep, camel, rabbit,goat, llama, and known as merino wool, Shetland wool, cashmere wool,alpaca wool, mohair etc. and includes wool fibers and animal hair. Themethod of the invention can be used on wool or animal hair material inthe form of top, fiber, yarn, or woven or knitted fabrics.

A Method of Cleaning Surfaces

A composition or bacteria culture of the invention may also be used forcleaning surfaces including hard and soft surfaces.

Thus, in a fourth aspect the invention relates to a method of cleaning asurface comprising subjecting said surface to a composition or bacterialculture of the invention.

Examples of contemplated hard surfaces are concrete, metal, glass,ceramic, plastic, linoleum and similar surfaces. Hard surfaces aretypically found in toilets, shower stalls, bathtubs, sinks, countertops,walls, floors and also include to road surfaces.

Examples of contemplated soft surfaces include carpet, furniture,upholstery fabric, slippers, clothing and other fibrous materialsurfaces.

It should be mentioned that compositions or bacterial cultures of theinvention are also contemplated for cleaning objects such as drains oroutlet pipes for waster water, sewers from, e.g., homes or industrialenterprises, vehicles, holding tanks, septic tanks etc. It is alsocontemplated using compositions or bacteria cultures of the inventionfor degrading, e.g., organic waste materials.

In a specifically contemplated embodiment the invention relates to amethod of cleaning carpets or other fibrous material surfaces.

It is to be understood that carpet cleaning cleans the carpet, but mayalso prevent or control odors from, e.g., organic spills, such as foodand the like.

The odor control may be preventive or precautionary, i.e., added to thecarpet, e.g., during manufacture of the carpet or fibrous material inquestion or after installation of a new carpet, or may also be used for,e.g., spot cleaning or full scale cleaning of soiled carpet or fibrousmaterials.

In a preferred embodiment of the invention the composition or culturefor cleaning surfaces, such as soft surfaces, especially carpets andother fibrous material, comprise the following strains alone or incombination: PTA-7548 and PTA-7547. The ratio between the two culturesmay be between 1:10 to 10:1, preferably 1:2 to 2:1, such as around 1:1.

The invention also relates to a method of preventing and/or controllingodor caused by organic material spilled on carpet or other fibrousmaterial, comprising applying a bacteria culture of the invention or acomposition of the invention to the carpet before and/or after spill oforganic material on the carpet or other fibrous material. The bacteriaculture is applied to the carpet at a concentration of between 10⁵ and10⁹, preferably between 10⁶ and 10⁸ cells per gram of carpet fiber,especially 10⁷ cells per grams of carpet fibers.

Use of Bacteria Culture of the Invention

In the final aspect, the invention relates to the use of a compositionor bacteria culture of the invention for cleaning or washing fabricand/or soft or hard surfaces. It is also contemplated to usecompositions or bacteria cultures of the invention for degrading, e.g.,organic waste materials. In a preferred embodiment a bacteria culture ofthe invention or a combination thereof, especially PTA-7548 andPTA-7547, are used in a carpet cleaner composition, especially onedisclosed in WO2007/076337 (which is hereby incorporated by reference).

The carpet cleaner may be a carpet extraction cleaner or a carpet spotcleaner. Examples of such carpet cleaners are disclosed in Examples 10and 11 below. In a preferred embodiment the bacteria cultures used inthe carpet cleaners are PTA-7548 and/or PTA-7547. It should beunderstood that the bacteria culture(s) should be present in effectiveamounts. Effective amounts can easily be determined by one skilled inthe art.

Materials & Methods Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty at American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20108, USA, and given the followingaccession number:

Identification Accession Number Date of Deposit Bacillusamyloliquefaciens PTA-7541 20 Apr. 2006 Bacillus amyloliquefaciensPTA-7542 20 Apr. 2006 Bacillus atrophaeus PTA-7543 20 Apr. 2006 Bacillusamyloliquefaciens PTA-7544 20 Apr. 2006 Bacillus amyloliquefaciensPTA-7545 20 Apr. 2006 Bacillus amyloliquefaciens PTA-7546 20 Apr. 2006Bacillus subtilis subsp. Subtilis PTA-7547 20 Apr. 2006 Bacillusvelezensis PTA-7548 20 Apr. 2006 Bacillus amyloiquefaciens PTA-7549 20Apr. 2006 Bacillus simplex PTA-7550 20 Apr. 2006 Bacillus simplexPTA-7789 18 Aug. 2006 Bacillus amyloliquefaciens PTA-7790 18 Aug. 2006Bacillus amyloliquefaciens PTA-7791 18 Aug. 2006 Bacillus atrophaeusPTA-7792 18 Aug. 2006 Bacillus amyloliquefaciens PTA-7793 18 Aug. 2006

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a pure culture of the deposited strain. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application or its progeny arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Fabrics:

All fabrics were purchased from Tesffabrics, Inc., West Pittson, Pa.18643, USA

Fabric Catalog Number Ground in clay on cotton STC GC C Synthetic Sebumon cotton STC SS DSC Ballast soil C-S-31 cotton soiled with aged bloodC-S-01 cotton soiled with butterfat and colorant C-S-10 cotton soiledwith oil, <60° C. C-09 soiled cotton with used motor oil W-10-GM

Fabric in Example 9 was obtained from Warwick Equest:

WARWICK EQUEST LIMITED Unit 55, Consett Business Park Consett, CountyDurham DH8 6BN ENGLAND Media and Reagents:

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

PCB (Plate Count Broth) purchased from Difco, Franklin Lakes, N.J., USA.LB (Luria-Bertani Broth) purchased from Difco, Franklin Lakes, N.J.,USA.

10D Sebum and particulates (carbon black) AS 12 Composite general soil(oil, milk protein, particulates) CS 62 Pork lard stained with sudan redHamburger grease stained with Macrolex Violet Dye.

Equipment Spectrophotometer: Gretag-Macbeth Color Eye 7000A MethodsFabric Stain Cleaning Procedure

An overnight culture of bacteria is grown in 10 ml in a complex nutrientrich media like PCB or LB at 35° C. with shaking at 250 rpm. Any culturethat does not reach a minimal OD₆₀₀ of 1.0 is re-inoculated at a laterdate and not used.

SSC₃ Minimal Media is used according to the following recipe:Base Media (all values in g/L unless otherwise noted)

NH₄Cl 0.8 MgSO₄ 0.2 CaCl₂•2H₂O 0.01 FeCl₃ 0.005 KH₂PO₄ 0.15 TraceMinerals 1 ml/L Glucose 2.0 MOPS 5.1 pH to 8.01000× Trace Minerals (all values in mg/L)

FeSO₄•7H₂O 28 ZnSO₄•7H₂O 140 MnSO₄•H₂O 84 CoCl₂•6H₂O 24 CuSO₄•5H₂O 25NaMoO₄•2H₂O 24

Microtiter plates containing punched stained fabrics are used as is. 200microliters of sterile SSC is added to every well.

5 microliters of the overnight culture is inoculated into the 200microliters of SSC₃ containing 0.2% glucose (w/v) that is added in theprevious step. Plate is grown with shaking at 35° C. for 48 hours. Aftergrowth, wells are rinsed with DI water 3×, then the fabric is driedovernight in a 35° C. incubator for photography

For shake flask studies:

10 ml overnight cultures of strains are grown in PCB at 35° C. withshaking at to 200 rpm. The next day, 0.25 ml of this culture is used toinoculate 10 ml of SSC₃ containing 0.2% glucose. This minimal mediaculture is also grown overnight at 35° C. with shaking at 200 rpm.

0.5 ml of this overnight culture is used to inoculate 150 ml of eachSSC₃ culture+stain fabric. Negative controls containing stained fabricwill be used containing 0.005% (w/v) myacide to inhibit all bacterialgrowth. Control fabric samples will be treated identically toexperimental samples. All culture flasks will be grown for 48 hours at35° C. with shaking at 200 rpm.

Fabric samples are removed, rinsed with distilled water and analyzed forreflective analysis on Gretag-Macbeth Color Eye 7000A spectrophotometer.The deltaE value is determined.

EXAMPLES Example 1 Cleaning of Blood Stained Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on cotton fabric soiled with aged blood (TestfabricsInc., PA, USA).

Strains: Identification DeltaE Cotton (control) 41.7 PTA-7547 Bacillussubtilis 22.81 PTA-7542 Bacillus amyloliquefaciens 28.32 PTA-7550Bacillus simplex 27.02 PTA-7548 Bacillus velezensis 33.84 PTA-7543Bacillus atrophaeus 23.92 PTA-7544 Bacillus amyloliquefaciens 20.67PTA-7545 Bacillus amyloliquefaciens 23.87 PTA-7546 Bacillusamyloliquefaciens 17.99 PTA-7549 Bacillus amyloliquefaciens 30.75

Example 2 Cleaning of Cotton Fabric with Ballast Stains

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on ballast soiled cotton fabric (Testfabrics Inc., PA,USA).

Strains: Identification DeltaE Cotton (Control) 20.3 PTA-7547 Bacillussubtilis 18.29 PTA-7542 Bacillus amyloliquefaciens 13.49 PTA-7550Bacillus simplex 13.34 PTA-7543 Bacillus atrophaeus 11.17 PTA-7545Bacillus amyloliquefaciens 9.78 PTA-7546 Bacillus amyloliquefaciens12.17 PTA-7549 Bacillus amyloliquefaciens 14.98 PTA-7792 Bacillusatrophaeus 16.51 PTA-7793 Bacillus amyloliquefaciens 6.51

Example 3 Cleaning of Butterfat Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on butterfat soiled cotton fabric (Testfabrics Inc.,PA, USA).

Strains: Identification DeltaE Cotton (Control) 15.7 PTA-7547 Bacillussubtilis 4.12 PTA-7542 Bacillus amyloliquefaciens PTA-7548 Bacillusvelezensis 4.87 PTA-7546 Bacillus amyloliquefaciens 5.45 PTA-7549Bacillus amyloliquefaciens 4.63

Example 4 Cleaning of Cooking Oil Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on cooking oil soiled cotton fabric (Testfabrics Inc.,PA, USA).

Key Strains: Identification DeltaE Cotton (Control) 18.7 PTA-7547Bacillus subtilis 5.15 PTA-7543 Bacillus atrophaeus 4.82 PTA-7544Bacillus amyloliquefaciens 3.26 PTA-7545 Bacillus amyloliquefaciens 2.96PTA-7541 Bacillus amyloliquefaciens 3.09

Example 5 Cleaning of Sebum Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on sebum soiled cotton fabric (Testfabrics Inc., PA,USA).

Key Strains: Identification DeltaE Cotton (Control) 19.3 PTA-7547Bacillus subtilis 4.17

Example 6 Cleaning of Sebum and Particulate Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on sebum and particulate soiled cotton fabric(Testfabrics Inc., PA, USA).

Key Strains: Identification DeltaE Cotton (Control) 18.3 PTA-7790Bacillus amyloliquefaciens 2.85 PTA-7792 Bacillus atrophaeus 3.47

Example 7 Cleaning of Composite General Soil Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on composite general soil soiled cotton fabric(Testfabrics Inc., PA, USA).

Key Strains: Identification DeltaE Cotton (Control) 18.3 PTA-7790Bacillus amyloliquefaciens 6.42

Example 8 Cleaning of Pork Lard Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on pork lard soiled cotton fabric (Testfabrics Inc.,PA, USA).

Key Strains: Identification DeltaE Cotton (Control) 26.33 PTA-7790Bacillus amyloliquefaciens 19.36 PTA-7789 Bacillus simplex 19.95

Example 9 Cleaning of Hamburger Grease Soiled Cotton Fabric

The following Bacillus strains deposited at ATCC were tested followingthe “Fabric stain cleaning procedure” described in the “Methods &Methods”-section on Hamburger grease soiled cotton fabric (WarwickEquest, Consett, England).

Key Strains: Identification DeltaE Cotton (Control) 21.18 PTA-7793Bacillus amyloliquefaciens 5.38 PTA-7791 Bacillus amyloliquefaciens 4.18

Example 10 Carpet Spot Removers

In each formulation, the active Sodium Octyl Sulfonate is introduced asB10-TERGE® PAS-8S (Stepan Company), which is a solution containing 37.8%active Sodium Octyl Sulfonate. In the following examples where SodiumOctyl Sulfonate is used, the quantity of Sodium Octyl Sulfonate is givenas percent actives.

A. Anionic Surfactant and Nonionic Surfactant in a Ratio of about 6:1(Formulation A).

This formulation is a starting formulation to be used as active cleaningbase in a carpet spot remover.

Material % By Weight Function Water Q.S. Solvent for all other materialsSodium Octyl 1.28 Water soluble anionic surfactant, Sulfonate allowspowdery residue Tomadol 91-6 0.23 Water soluble nonionic surfactantIsopropyl Alcohol 2.50 Organic solvent to help with water- insolublestain removal. Kathon CG/ICP 0.050 Preservative Bronopol 0.025Preservative (BIOBAN ™ BP-PLUS) Citric Acid 0.25 Provide buffering pH6-7 Caustic Soda 0.30 pH adjustment of citric acid to pH 6-7 Bacteriacultures 5.4 × 10⁸ cfu/ml cleaning and odor controlling PTA-7548 andingredient PTA-7547

B. 50/50 Tomadol 91-6/Tomadol 91-2.5, 1.50% Total Surfactant(Formulation B)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.28 Tomadol 91-60.11 Tomadol 91-2.5 0.11 Isopropyl Alcohol 2.50 Kathon CG/ICP 0.050Bronopol (BIOBAN ™ BP-PLUS) 0.025 Citric Acid 0.25 Caustic Soda 0.30Bacteria cultures 5.4 × 10⁸ cfu/ml PTA-7548 and PTA-7547

C. 30/70 Tomadol 91-6/Tomadol 91-2.5, 1.51% Total Surfactant(Formulation C)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.28 Tomadol 91-60.07 Tomadol 91-2.5 0.16 Isopropyl Alcohol 2.50 Kathon CG/ICP 0.050Bronopol (BIOBAN ™ BP-PLUS) 0.025 Citric Acid 0.25 Caustic Soda 0.30Bacteria cultures 5.4 × 10⁸ cfu/ml PTA-7548 and PTA-7547

D. No Isopropyl Alcohol, 2.30% Total Surfactant (Formulation D)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.96 Tomadol 91-60.10 Tomadol 91-2.5 0.24 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™ BP-PLUS)0.025 Citric Acid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4 × 10⁸cfu/ml PTA-7548 and PTA-7547

D1. 0/100 Tomadol 91-6/Tomadol 91-2.5, 2.31% Total Surfactant(Formulation D1)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.96 Tomadol91-2.5 0.35 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™ BP-PLUS) 0.025 CitricAcid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4 × 10⁸ cfu/ml PTA-7548and PTA-7547

E. 20/80 Tomadol 91-6/Tomadol 91-2.5, 1.60% Total Surfactant(Formulation E)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.36 Tomadol 91-60.05 Tomadol 91-2.5 0.19 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™ BP-PLUS)0.025 Citric Acid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4 × 10⁸cfu/ml PTA-7548 and PTA-7547

F. 20/80 Tomadol 91-6/Tomadol 91-2.5, 1.80% Total Surfactant(Formulation F)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.53 Tomadol 91-60.054 Tomadol 91-2.5 0.216 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™BP-PLUS) 0.025 Citric Acid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4× 10⁸ cfu/ml PTA-7548 and PTA-7547

G. 20/80 Tomadol 91-6/Tomadol 91-2.5, 1.90% Total Surfactant(Formulation G)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.62 Tomadol 91-60.057 Tomadol 91-2.5 0.228 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™BP-PLUS) 0.025 Citric Acid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4× 10⁸ cfu/ml PTA-7548 and PTA-7547

H. 20/80 Tomadol 91-6/Tomadol 91-2.5, 2.00% Total Surfactant(Formulation H)

Material % By Weight Water Q.S. Sodium Octyl Sulfonate 1.70 Tomadol 91-60.06 Tomadol 91-2.5 0.24 Kathon CG/ICP 0.050 Bronopol (BIOBAN ™ BP-PLUS)0.025 Citric Acid 0.25 Caustic Soda 0.30 Bacteria cultures 5.4 × 10⁸cfu/ml PTA-7548 and PTA-7547

Example 11 Carpet Extraction Cleaner

An aqueous cleaning composition for use in carpet extraction cleaning isdescribed below. The cleaning compositions illustrate products that theconsumer purchases and dilutes in water by adding 2 ounces (56.7 grams)to the filling tank and filling with hot water to make a total of onegallon (3.79 liters).

Five cleaning composition formulations in weight/weight percentage aregiven in the table below. The ratio of TOMADOL 91-6 to TOMADOL 91-2.5 isalso given as a percentage ratio of the total content of TOMADOL 91-6and TOMADOL 91-2.5. Note that for all of these formulations, the onlychange is the relative amounts of TOMADOL 91-6 and TOMADOL 91-2.5. Thesein-use cleaning solution are prepared by adding 6.25 g of the cleaningformulations to a bottle, and bringing the total mass to 400 g with tapwater.

50/50 0/100 25/75 15/85 20/80 Water Q.S. Q.S. Q.S. Q.S. Q.S. SodiumOctyl 2.34 2.34 2.34 2.34 2.34 Sulfonate Tomadol 91-6 0.96 0.00 0.480.29 0.38 Tomadol 91-2.5 0.96 1.91 1.43 1.63 1.53 Kathon 0.050 0.0500.050 0.050 0.050 Bronopol 0.025 0.025 0.025 0.025 0.025 Citric Acid4.25 4.25 4.25 4.25 4.25 Caustic Soda 4.90 4.90 4.90 4.90 4.90 Bacteriacultures 5.4 × 5.4 × 5.4 × 5.4 × 5.4 × PTA-7548 and 10⁸ cfu/ 10⁸ cfu/10⁸ cfu/ml 10⁸ cfu/ml 10⁸ cfu/ml PTA-7547 ml ml

Cleaning composition formulations. The ratio of Tomadol 91-6 to Tomadol91-2.5 is also given as a percentage ratio of the total content ofTomadol 91-6 and Tomadol 91-2.5.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1: A method for treating soils or stains on a surface comprisingsubjecting the soil or stain to one or more isolated bacterial strainsselected from the group consisting of: the strain having the depositaccession number PTA-7541; the strain having the deposit accessionnumber PTA-7542; the strain having the deposit accession numberPTA-7543; the strain having the deposit accession number PTA-7544; thestrain having the deposit accession number PTA-7545; the strain havingthe deposit accession number PTA-7546; the strain having the depositaccession number PTA-7547; the strain having the deposit accessionnumber PTA-7548; the strain having the deposit accession numberPTA-7549. the strain having the deposit accession number PTA-7550, thestrain having the deposit accession number PTA-7789, the strain havingthe deposit accession number PTA-7790, the strain having the depositaccession number PTA-7791, the strain having the deposit accessionnumber PTA-7792, the strain having the deposit accession numberPTA-7793, or a mixture of two or more of the strains. 2: The method ofclaim 1, wherein the method further comprises subjecting the soil orstain to one or more enzymes. 3: The method of claim 2, wherein the oneor more enzymes are selected from the group consisting of a protease, analpha-amylase, a cellulase, a lipase, a peroxidase, an oxidase, apectate lyase, a mannase, or mixtures thereof. 4: The method of claim 1,wherein the one or more bacterial strains is the strain having thedeposit accession number PTA-7541. 5: The method of claim 1, wherein theone or more bacterial strains is the strain having the deposit accessionnumber PTA-7542. 6: The method of claim 1, wherein the one or morebacterial strains is the strain having the deposit accession numberPTA-7543. 7: The method of claim 1, wherein the one or more bacterialstrains is the strain having the deposit accession number PTA-7544. 8:The method of claim 1, wherein the one or more bacterial strains is thestrain having the deposit accession number PTA-7545. 9: The method ofclaim 1, wherein the one or more bacterial strains is the strain havingthe deposit accession number PTA-7546. 10: The method of claim 1,wherein the one or more bacterial strains is the strain having thedeposit accession number PTA-7547. 11: The method of claim 1, whereinthe one or more bacterial strains is the strain having the depositaccession number PTA-7548. 12: The method of claim 1, wherein the one ormore bacterial strains is the strain having the deposit accession numberPTA-7549. 13: The method of claim 1, wherein the one or more bacterialstrains is the strain having the deposit accession number PTA-7550. 14:The method of claim 1, wherein the one or more bacterial strains is thestrain having the deposit accession number PTA-7789. 15: The method ofclaim 1, wherein the one or more bacterial strains is the strain havingthe deposit accession number PTA-7790. 16: The method of claim 1,wherein the one or more bacterial strains is the strain having thedeposit accession number PTA-7791. 17: The method of claim 1, whereinthe one or more bacterial strains is the strain having the depositaccession number PTA-7792. 18: The method of claim 1, wherein the one ormore bacterial strains is the strain having the deposit accession numberPTA-7793. 19: The method of claim 1, wherein the surface is a softsurface or a hard surface. 20: The method of claim 19, wherein the softsurface is a laundry or fabric.